Epigenetic determinants of gene expression are fundamentally important to innumerable pathophysiologic processes including oncogenesis. CpG island DNA methylation is a common epigenetic modification of particular interest. In glioblastoma (GBM), the most common and aggressive adult brain tumor, hundreds of genes are subjected to DNA hypermethylation at promoter CpG islands; and tumor methylation status has been found to define distinct GBM tumor subsets and predict clinical behavior. Therefore, dissecting the transcriptional consequences of CpG island methylation is critically important to understanding cancer biology and translating epigenetic mechanisms to clinical advantage. The classical dogma states that promoter CpG island methylation influences cancer biology by silencing tumor suppressor genes through sequence- independent manner. However, this dogma is challenged by our recent studies, which indicate that some transcription factors (TFs), including kruppel-like factor 4 (KLF4), preferentially bind to methyl CpG (mCpG)- containing motifs, thereby transactivating gene expression. The zinc finger TF KLF4 is a cancer driver gene shown to be up-regulated in GBMs. Using a site-specific KLF4 mutant lacking the ability to bind to methylated DNAs, we found that mCpG-dependent KLF4 binding promotes GBM cell migration via the transactivation of genes in cell motility signaling pathways, including the small GTPase RhoC and calpain small subunit 1 (CAPNS1). We further demonstrate that histone modifications are involved in KLF4-mCpG-mediated gene transactivation. In this proposal, we will test our hypothesis that promoter mCpG plays an active role in gene regulation by directing TF binding, histone modifications and gene activation, thereby influencing cancer cell phenotypes. In Aim 1, we will determine if KLF4-mCpG interactions regulate GBM stem cell migration and self- renewal in vitro and in vivo. In Aim 2, we will use complementary approaches to rigorously validate this new mechanism by which KLF4 activates cell motility genes and promotes GBM cell migration via its ability to bind to mCpG-containing promoters. We will perturb KLF4-mCpG interactions in GBM cells using genome editing methodology to act upon both the cis- and trans-regulatory elements. In Aim 3, we will use unbiased approaches (ChIP-sequencing and RNA-sequencing) to identify the global transcriptional networks directly regulated by KLF4-mCpG interactions in GBM stem cells. This will be the first comprehensive study to assess how DNA methylation and TF interactions transactivate gene expression and influence tumor cell behaviors. Positive results will be paradigm shifting and substantially expand current knowledge regarding how DNA modification regulates gene expression and influences oncogenesis. Dissecting the biological consequences of KLF4-mCpG interactions in GBMs and ultimately extending to other TFs and other cancer subtypes will revolutionize our understanding of how epigenetic modifications contribute to oncogenesis.